The long-term objective of the research is to significantly advance our understanding of nucleic acid structure, thermodynamics, and kinetics. The immediate objective of the proposed research is to significantly enhance the experimental and theoretical capability for the study of RNA folding dynamics, which are inherently linked to RNA function. RNA folding is not well understood despite RNA<s pivotal role in numerous biological functions such as catalysivs (ribozymes) and gene expression silencing (RNA interference, RNAi). RNA has direct medical relevance as shown by RNAi therapies currently in Phase III clinical trials for eye disease (age-related macular degeneration or AMD) and in Phase II trials for viral infections (respiratory syncytial virus or RSV). The proposed research involves the development (synthesis, characterization) and application of multi-spectroscopic, site-specific (MS3) probes to the study of local environments in DNA and RNA structure. The probes are nitrile- and azide-derivatized nucleosides. The nitrile (CN) and azide (N3) groups represent small perturbations to the structure of the nucleosides, while providing sensitive IR probes of local nucleic acid environments. Upon 15N labeling of the terminal nitrogen atom of CN and N3, 15N NMR spectroscopy can be used to probe local nucleic acid environments. Overall, the MS3 probes have the unique ability to provide site-specific information about all three major environments in nucleic acids: the major groove, the minor groove, and the sugar/backbone region, via two powerful spectroscopic techniques: infrared (IR) and 15N NMR spectroscopy. The MS3 probes will be synthesized either by using either literature procedures or by new synthetic routes utilizing azo transfer reactions. The spectral dependence of the CN and N3 stretching frequencies and 15N NMR chemical shifts on solvent (hydration), base pair formation, and temperature will be investigated. Subsequently, the MS3 probes will be incorporated at various positions in a 12 base pair DNA oligomer and an 8 nucleotide RNA hairpin. These nucleic acid structures will be investigated by equilibrium temperature-dependent IR and 15N NMR spectroscopic techniques to probe the various local environments present in these structures upon melting. Conformational changes induced by drug binding will also be explored in the DNA oligomer using the MS3 probes. These results will allow the parameterization and validation of force fields suitable for nucleic acids by our collaborators (Professors Corcelli and MacKerell) to be performed. Another goal of this health related research is to train eight undergraduate students in organic synthesis and spectroscopic data acquisition and analysis techniques. PUBLIC HEALTH RELEVANCE: The proposed research centers on significantly enhancing the capability to study RNA folding dynamics, which is inherently linked to RNA function, through the development of multi-spectroscopic, site- specific (MS3) probes. RNA folding is not well understood despite RNA<s pivotal role in numerous biological functions such as catalysis (ribozymes) and gene expression silencing (RNA interference, RNAi). RNA has direct medical relevance as shown by RNAi therapies currently in Phase III clinical trials for eye disease (age-related macular degeneration or AMD) and in Phase II trials for viral infections (respiratory syncytial virus or RSV).